This project is concerned with the functional roles of the sensory arrestin genes, arr1 and arr2, in the regulation of dipteran olfactory signal transduction. Specifically, I have examined the sensory arrestins of the fruit fly, Drosophila melanogaster, and the malaria vector mosquito, Anopheles gambiae (An. gambiae). I have demonstrated that transgenic expression of An. gambiae arr1 in the antennae of arr1 mutant fruit flies sufficiently rescues olfactory deficits in these mutant flies. This implies functional orthology between the fruit fly and mosquito arr1 homologues. I also sought to examine spatial and temporal characteristics of these genes in the antennae of D. melanogaster. I attempted to identify the spatial expression patterns of arr1 and arr2 mRNA in antennal neurons via fluorescence in situ hybridization. However, the expression levels of these genes lie below the threshold of detection for the applied methodology, as no signal corresponding to the expression of these genes was detected. I utilized time-controlled induction of wild-type arr1 transgene paradigms in arr1 mutant fruit flies to assess the role of this gene as it pertains to the temporal dynamics of the effects of this gene on the olfactory system of the fly. The results of these studies are inconclusive and require further experimentation.

Common diseases with a genetic basis are likely to have a very complex etiology, in which the mapping between genotype and phenotype is far from straightforward. A new comprehensive statistical and computational strategy for identifying the missing link between genotype and phenotype is proposed, which emphasizes the need to address heterogeneity in the first stage of any analysis. A simulation study comparing three unsupervised clustering methods was conducted, and the best methodBayesian Classificationwas evaluated further for its performance and applicability to real data under a wide range of simulation conditions. <p> The proposed two-stage analysis strategy was then applied to late-onset Alzheimer disease data. Bayesian Classification found statistically significant clusterings for independent family-based and case-control datasets, which used the same five markers in LRRTM3 as their most influential in determining cluster assignment. In subsequent analyses to detect main effects and gene-gene interactions, markers in four genesPLAU, IDE, CDC2 and ACEwere found to be associated with late-onset Alzheimer disease in particular subsets of the data based on their LRRTM3 haplotype. While each of these genes are viable candidates for LOAD based on their known biological function, further studies are needed to replicate these statistical findings and to elucidate possible biological interaction mechanisms between LRRTM3 and these genes. <p> Going forward, genetic studies will increasingly focus time and resources to collecting phenotypic data that can refine definitions or subcategories of traits or diseases and can serve as endophenotypes, which are more likely to have simple etiologies and to directly map to specific genetic markers. In the case of neurological diseases, one collection of phenotyping technologies which has matured considerably over the past five to ten years is neuroimaging. In addition, an emphasis on possible biological mechanisms of disease has positively influenced the design of behavioral assessment tools, increasing their utility as phenotyping tools, which provide endophenotypes that can be mapped to genotypic data. Methodologies enabling the integration of disparate data sources (genotyping and neuroimaging or behavioral) must be investigated in order to harness the power inherit in their complexity.

NEUROSCIENCE ROLE OF α2A ADRENERGIC RECEPTORS IN EXTINCTION OF POSITIVE AND NEGATIVE VALENCE LEARNED BEHAVIORS Adeola R. Davis Dissertation under the direction of Professor Danny Winder The work presented in this dissertation focuses on the role of α2 adrenergic receptors in extinction of positive and negative valence learned behaviors as well as investigating the mechanism of the action of a pharmaceutical agent, yohimbine. Mechanisms underlying extinction of positive valence associations such as drug self administration and place preference are poorly understood, yet may have important relevance to addiction treatment. Data suggest a major role for the noradrenergic system in extinction of fear-based learning. Administration of yohimbine, an α2 adrenergic receptor antagonist, facilitates extinction of learned fear. Employing a combination of pharmacological and genetic approaches, we investigated the role of the α2 adrenergic receptor in extinction of cocaine-induced conditioned place preference and lithium chloride-induced conditioned place aversion. Additionally, yohimbine is a commonly used pharmaceutical agent and the mechanism of action is often attributed to solely antagonism at the α2 adrenergic receptors. However, we investigate potential off target sites using electrophysiological methods. In this research, the results indicate that extinction of conditioned place preference with yohimbine occurs independently of the α2 adrenergic receptors although the receptors modify the effect. Further, extinction with yohimbine of conditioned place preference and conditioned place aversion similarly impairs extinction of the behaviors. Lastly, we found a novel target for extinction behaviors with the use of electrophysiology.

Dissertation under the direction of Professor Edwin J. WEeber <p> Apolipoprotein E (apoE) interacts with lipoprotein receptors in the hippocampus. These receptors are intimately involved in the modulation of synaptic plasticity, learning and memory. Isoform variation in apoE may differentially alter this lipoprotein receptor-dependent synaptic modulation as apoE isoforms are associated with human neurodegenerative disorders. In the present study, apoE2, apoE3 and apoE4 targeted replacement (TR) mice were used along with recombinant human apoE isoforms to determine the role of apoE isoforms in hippocampus area CA1 synaptic function. While synaptic transmission is unaffected by apoE isoform, long-term potentiation (LTP) is significantly enhanced in apoE4 TR mice versus apoE2 TR mice. ApoE isoform-dependent differences in LTP induction require NMDA-receptor function, and apoE isoform expression alters activation of both ERK1/2 and JNK1/2. Acute application of apoE isoforms similarly alters LTP induction (E4>E2) but both apoE2 and apoE4 decrease NMDA receptor mediated field potentials. Furthermore, acute apoE isoform application does not alter ERK1/2 and JNK1/2 activation like chronic apoE expression. <p> ApoE isoforms also differentially alter learning and memory in adult male mice. ApoE TR animals were utilized to isolate the behavioral effects of apoE isoform. ApoE-deficient and C57BL/6J (wild-type) mice were tested concurrently, evaluating all in working memory, associative learning, and spatial memory. General locomotion, sensorimotor gating, and working memory were not altered by either the lack of murine apoE or the presence of human apoE isoforms. Testing of long-term contextual associative memory revealed significant differences only between apoE2 and apoE4 TR animals. ApoE4 TR animals were impaired in spatial learning while apoE3 TR animals were not. <p> These findings demonstrate specific, isoform-dependent effects of human apoE isoforms on adult hippocampus synaptic plasticity. There may be mechanistic differences between chronic apoE isoform expression and acute apoE isoform exposure in this modulation. These findings also support the hypothesis that apoE isoforms play a specific role in modulating learning and memory. <p>

How does the brain select visual targets for eye movements? We recorded neural activity while macaques performed visual search in which they were trained to move their eyes to a target stimulus among an array of distractor stimuli for a reward. Three signals were used to measure the relationship between the decision to move the eyes to a target and neural activity: spike rates from neurons in the frontal eye field (FEF), local field potentials from FEF, and event-related potentials recorded from the skull. We found that (1) FEF neurons cooperated and competed to select visual targets, measured using correlations between spike times of simultaneously recorded neurons, (2) FEF neurons interacted more when the visual search task was easier (i.e., the target and distractors were easily discriminable), measured using a multivariate analysis and decreased firing variability before eye movements, (3) FEF neurons decreased firing variability around the time of target selection by the mean firing rate, (4) FEF neurons were distinguished both functionally and biophysically, on the basis of action potential width, (5) FEF selected search targets from distractors before the macaque homologue of a human cognitive event-related potential (the N2pc) that marks the allocation of attention, and (6) the time between visual search array presentation and the time that FEF neurons discriminated between target and distractors was later when there were more stimuli the animal needed to choose from.

Transcripts encoding 5HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Due to a surprising lack of ligand affinity data in mice, we first established the selectivity of several 5-HT2 receptor ligands in mice. Using mice solely expressing the 5HT2C-VGV (VGV/Y) receptor, we demonstrate reduced G-protein coupling efficiencies and high-affinity agonist binding of brain 5HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5HT2C receptor agonist was also seen in mice expressing 5HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5HT2C-VGV receptors had enhanced sensitivity to 5HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by massive increases in receptor binding sites in the brains of mice solely expressing 5HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in receptor binding site density. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall receptor functionality.

ABSTRACT Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder predominantly afflicting the striatum. It is clear that an expansion of a glutamine encoding CAG triplet-repeat in the Huntingtin (HTT) gene causes HD. However, the molecular basis of the selective pathology has not been elucidated. The wild-type HD protein has recently been discovered to play a role in iron homeostasis and associates with copper. Like HD, copper (Cu), iron (Fe), and manganese (Mn) neurotoxicity is associated with basal ganglia dysfunction. Environmental overexposure to the essential metal Mn leads to accumulation of Mn in the basal ganglia and a parkinsonian-like condition called manganism. Recognizing the pathophysiological similarities between HD and the neurotoxicity of these metals, we hypothesized that metals may exhibit gene-environment interactions with the HD gene, HTT. To determine the contribution of specific physiological and pathological processes to selective neuropathology in HD, we tested various metals that influence similar neuronal populations to identify modifications in cellular functions of the normal or disease-causing HD protein. Using a cellular model of HD, we have found that expression of the mutant HTT protein induces a resistance specifically to Mn toxicity. To understand the cellular basis of this phenotype, we investigated the possibility that HTT may alter Mn transport. We found that net accumulation of Mn is substantially decreased in cells expressing mutant HTT under standard culture conditions and after Mn exposure. Given previous reports linking HTT with iron homeostasis, we examined a role for Fe transport in this phenotype. Our data indicate that functional differences in Fe homeostasis only partially contribute to the Mn transport deficit. Assessment of other known Mn transporters (e.g. Divalent metal transporter 1 (DMT1) and the Zip8 family) failed to reveal a significant contribution for these pathways. To corroborate the Mn transport defects in vivo, we exposed the YAC128Q mouse model of HD to Mn and found that mutant HTT selectively impairs net Mn transport in the striatum, the brain region most vulnerable to HD. Therefore, we conclude that mutant HTT alters Mn homeostatic control and has the potential to contribute to selective degeneration.

We studied the modulation of ¦Á4¦Â3¦Ã2L and ¦Á4¦Â3¦Ä GABAA receptor currents by two protein kinases, PKA and PKC. Although modulation of synaptic ¦Á1¦Â¦Ã2 GABAA receptor isoforms has been studied widely, study of the modulation of peri- and extrasynaptic ¦Á¦Â¦Ä and non-synaptic ¦Á¦Â¦Ã GABAA receptors by protein phosphorylation is lacking. Using patch-clamp recording, we compared the effects of protein phosphorylation of ¦Á4¦Â3¦Ã2L and ¦Á4¦Â3¦Ä GABAA receptors under different levels of activation that included spontaneous openings, tonic currents activated by low GABA concentrations and phasic currents activated by high GABA concentrations. We found that PKA-activation preferentially increased spontaneous ¦Á4¦Â3¦Ä receptor currents by increasing single channel open frequency and decreased GABA-activated steady-state, tonic ¦Á4¦Â3¦Ä currents, but only had small effects on spontaneous and GABA-activated tonic ¦Á4¦Â3¦Ã2L currents, indicating the differential modulation of tonic inhibition mediated by ¦Á4¦Â3¦Ã2L and ¦Á4¦Â3¦Ä GABAA receptors. In contrast, both PKA and PKC had similar effects on desensitization of phasic ¦Á4¦Â3¦Ã2L and ¦Á4¦Â3¦Ä currents, implying a common modulatory mechanism of protein phosphorylation in regulating synaptic current kinetics. Our study suggested that protein phosphorylation had profound effects on different GABAA receptor isoforms, which should also be determined by receptor sub-cellular (synaptic or non-synaptic) localization and by the level of ambient GABA (spontaneous or GABA-activated), thus ensuring precise modulation of specific GABAA receptor properties in specific brain areas.

GABA-A receptors are ligand-gated chloride channels that mediate the majority of fast inhibitory signaling in the central nervous system. Their kinetic properties determine the charge transfer and timing of inhibitory post-synaptic currents (IPSCs), and consequently, alterations in GABA-A receptor kinetics due to mutations, allosteric modulators, or post-translational modifications can significantly influence the function of neuronal circuits. However, one of the most defining kinetic features of GABA-A receptor currents their ability to undergo extensive and multi-phasic desensitization in the continued presence of agonist remains poorly understood. Indeed, while the phenomenon of desensitization has been well characterized, neither its microscopic kinetic basis nor its physiological relevance is clear. Many investigators have actually dismissed the phenomenon as an experimental artifact, as its visualization requires receptor activation for physiologically irrelevant durations. In the studies described herein, we challenge this notion, and argue instead that GABA-A receptor desensitization is a critical determinant of IPSC kinetics. Using a combination of patch-clamp electrophysiology and Markov modeling of GABA-A receptor function, we demonstrate that in addition to shaping the decay of individual IPSCs, desensitization is required for the phenomenon of repeated pulse inhibition, the loss of IPSC amplitude observed in the setting of high frequency stimulation. Interestingly, our results also suggest that desensitization is important for extrasynaptic signaling, where receptors are persistently activated by low concentrations of ambient GABA. Analytic solutions of Markov model equilibrium and non-equilibrium state occupancies demonstrate that the underlying desensitized state not only increases receptor affinity for GABA, but also buffers equilibrium currents from the effects of negative modulators and fluctuations in the ambient GABA concentration. Of note, the analytic solutions also exposed the microscopic kinetic determinants of desensitization, demonstrating a previously unrecognized dependence of this phenomenon on a subset of rate constants.

The brains ability to bind incoming auditory and visual stimuli depends critically on the temporal structure of this information. Specifically, there exists a window of time within which stimuli are highly likely to be bound together and perceived as part of the same environmental event. Several studies have characterized the temporal bounds of this window, but few have investigated its malleability. Here, the plasticity in the size of this temporal binding window was investigated using a perceptual learning paradigm in which participants were given feedback during a two-alternative forced choice (2-AFC) audiovisual simultaneity judgment task. Training resulted in a marked (i.e., approximately 40%) narrowing in the size of the window. To rule out the possibility that this narrowing was the result of changes in cognitive biases, a second experiment using a two-interval forced choice (2-IFC) paradigm was undertaken during which participants were instructed to identify a simultaneously presented audiovisual pair presented within one of two intervals. The 2-IFC paradigm resulted in a narrowing that was similar in both degree and dynamics to that using the 2-AFC approach. In a follow-up neruoimaging study, we sought to reveal the neural substrates of these changes. Eleven subjects completed a two-day 2-IFC audiovisual simultaneity judgment training paradigm, immediately before and after which they performed the same task during an event-related 3T fMRI session. A central zone of multisensory convergence, the posterior superior temporal sulcus (pSTS), as well as areas of auditory and visual cortex, exhibited robust BOLD decreases following training, and resting state and effective connectivity analyses revealed significant increases in functional coupling between these cortices after training. These imaging data represent the first evidence of the neural correlates underlying plastic change in adult multisensory networks that likely represent the substrate for a multisensory temporal binding window. These findings suggest a high degree of flexibility in multisensory temporal processing and have important implications for interventional strategies that may be used to ameliorate clinical conditions (e.g., autism, dyslexia) in which multisensory temporal function may be impaired.